<i>deoP1</i> promoter and operator mutants in <i>Escherichia coli</i>: isolation and characterization

Dandanel, G.; Hammer, Karin

doi:10.1111/j.1365-2958.1991.tb02083.x

Cited by 6 publications

(4 citation statements)

References 21 publications

(10 reference statements)

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“…This element is only weakly conserved among all promoters, but it has been noted for several promoters that lack clear -35 sequence homology (11,35,49,50). The element is not specifically required for activated promoters, since it is necessary in a CII-independent mutant (35) and for CRP-independent transcription of galPl (8,50).…”

Section: Resultsmentioning

confidence: 99%

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Sequence elements in the Escherichia coli araFGH promoter

1992

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The Eschcrichia coli araFGH operon codes for proteins involved in the L-arabinose high-affinity transport system. Transcriptional regulation of the operon was studied by creating point mutations and deletions in the control region cloned into a GalK expression vector. The transcription start site was confirmed by RNA sequencing of transcripts. The sequences essential for polymerase function were localized by deletions and point mutations. Surprisingly, only a weak -10 consensus sequence, and no -35 sequence is required. Mutation of a guanosine at position -12 greatly reduced promoter activity, which suggests important polymerase interactions with DNA between the usual -10 and -35 positions. A double mutation toward the consensus in the -10 region was required to create a promoter capable of significant AraC-independent transcription. These results show that the araFGH promoter structure is similar to that of the galPI promoter and is substantially different from that of the araBAD promoter. The effects of 11 mutations within the DNA region thought to bind the cyclic AMP receptor protein correlate well with the CRP consensus binding sequence and confirm that this region is responsible for cyclic AMP regulation. Deletion of the AraC binding site nearest the promoter, araFGl, eliminates arabinose regulation, whereas deletion of the upstream AraC binding site, araFG2, has only a slight effect on promoter activity.The RNA polymerase binding sites of many promoters have been studied in detail. A consensus sequence for recognition by RNA polymerase c7o includes two conserved hexanucleotide sequences located 10 and 35 bp upstream from the transcription start site (25,26,46,67). Additional sequences between and upstream of these elements are weakly conserved but can significantly affect promoter strength (2, 50). A model of polymerase recognition based on genetic experiments showing interactions of different amino acid motifs within the cr subunit with specific base pairs of the -10 and -35 regions has been developed (18,60,65). Our present knowledge of promoter structure is based primarily on the analysis of strong promoters that do not require accessory proteins. Promoters requiring activators frequently have a -35 region with poor similarity to the consensus (25). It is not clear, however, whether these promoters are simply characterized by a poor polymerase recognition site or whether they contain unique recognition elements. We are studying the Escherichia coli promoters that regulate genes involved in arabinose metabolism to understand structure-function relationships of promoters that require multiple activators for transcription.T`wo distinct systems are capable of active transport of the sugar L-arabinose in E. coli. A low-affinity, high-capacity symporter system is the product of the araE operon (6, 36, 62). At present only one protein is known to be encoded by this operon. A high-affinity system is encoded by the araFGH operon located at 41.9 min (2,000 bp) on the E. cdli map (9, 27, 37, 58). The product of th...

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Section: Resultsmentioning

confidence: 99%

“…The G residue has been shown to be important for expression from several promoters that require activator proteins (11,35,49 The gal and araF promoters both lack identifiable -35 sequences. PamaF matches the consensus at only one of six positions with any spacing from 15 to 19 bp upstream of the -10 hexamer.…”

Section: Resultsmentioning

confidence: 99%

Sequence elements in the Escherichia coli araFGH promoter

1992

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“…Plasmids pKCBIOI and pKCBiO2 were obtained by ligating H/ndll-digested plvlK112 into the unique Smal site in the multiple cloning site of the galK expression vector pGD52 (Dandanell and Hammer, 1991). Plasmid pMK112 contains a HindW site in the multiple cloning site immediately next to the Bgt\\-Xba\ junction.…”

Section: Recombinant Dna Techniques and Plasmid Constructionsmentioning

confidence: 99%

Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase

Danielsen

Kilstrup

Barilla

et al. 1992

Molecular Microbiology

123

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The nucleotide sequence of a 3.1 kb segment carrying the cytosine deaminase gene (codA) from Escherichia coli was determined. The sequence revealed the presence of two open reading frames, the first (codB) specifying a highly hydrophobic polypeptide and the second specifying cytosine deaminase. A two-codon overlap between the two reading frames indicates that they constitute an operon. Transcription of the operon was found to be regulated by exogenous purines. Polypeptides specified by each of the two reading frames were expressed in minicells, and the codB gene product was found to be highly enriched in the membrane fraction. Uptake experiments showed that the CodB protein is required for cytosine transport into the cell and that the intracellular accumulation of cytosine correlated with the codB gene dose. A topological model for the cytosine permease in the cytoplasmic membrane is proposed.

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“…In the deoCABD operon of Escherichia coli the DeoR repressor can bind to three operators OE, 01 and 02 located -886, -607 and -8 bp respectively, upstream of the transcription start of the deoP2 promoter (5,9,10). The deoR operator sites 01 and 02 which consist of a 16 bp palindromic sequence, overlap the -10 region of deoPI and deoP2, respectively (5,11,12,13).…”

Section: Introductionmentioning

confidence: 99%

DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology

Dandanell

1992

Nucl Acids Res

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The interoperator distance between a synthetic operator Os and the deoP2O2-galK fusion was varied between 46 and 176 bp. The repression of the deoP2 directed galK expression as a function of the interoperator distance (center-to-center) was measured in vivo in a single-copy system. The results show that the DeoR repressor efficiently can repress transcription at all the interoperator distances tested. The degree of repression depends very little on the spacing between the operators, however, a weak periodic dependency of 8-11 bp may exist.

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deoP1 promoter and operator mutants in Escherichia coli: isolation and characterization

Cited by 6 publications

References 21 publications

Sequence elements in the Escherichia coli araFGH promoter

Sequence elements in the Escherichia coli araFGH promoter

Characterization of the Escherichia coli codBA operon encoding cytosine permease and cytosine deaminase

DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology

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